Abstract

Otitis media (OM), characterized by the presence of mucus overproduction and excess inflammation in the middle ear, is the most common childhood infection. Nontypeable Haemophilus influenzae (NTHi) pathogen is responsible for approximately one-third of episodes of bacteria-caused OM. Current treatments for bacterial OM rely on the systemic use of antibiotics, which often leads to the emergence of multidrug resistant bacterial strains. Therefore there is an urgent need for developing alternative therapies strategies for controlling mucus overproduction in OM. MUC5AC mucin has been shown to play a critical role in the pathogenesis of OM. Here we show that curcumin derived from Curcuma longa plant is a potent inhibitor of NTHi-induced MUC5AC mucin expression in middle ear epithelial cells. Curcumin inhibited MUC5AC expression by suppressing activation of p38 MAPK by upregulating MAPK phosphatase MKP-1. Thus, our study identified curcumin as a potential therapeutic for inhibiting mucin overproduction in OM by upregulating MKP-1, a known negative regulator of inflammation.

1. Introduction

Mucin glycoproteins are a major component of mucus secretions in the middle ear, trachea, and digestive and reproductive tracts. Mucus production represents a protective innate defense mechanism to protect and lubricate the epithelium and trap invading pathogens for removal by the mucociliary clearance system [1]. However, in chronic infections, excess mucin impairs the mucociliary clearance system, resulting in mucus accumulation and poor function of the mucus-lined epithelial tracts. Of the ~24 mucin genes identified till date MUC5AC mucin has been shown to play a critical role in the pathogenesis of upper respiratory tract infections including otitis media (OM). OM is characteristic of the presence of mucus overproduction and excess inflammation in the middle ear [2, 3]. In patients with OM, increased mucus effusion into the ear’s tympanic cavity impairs the movement of the eardrum and middle ear bones and leads to hearing problems. A higher concentration of mucin in the middle ear effusion has been shown to correlate with the extent of hearing impairment [4, 5]. While mucin upregulation is an important innate defense response of the host to infections in the middle ear, excess mucin can lead to impaired mucociliary clearance and conductive hearing loss [2]. Therefore, mucin expression must be tightly controlled.

Nontypeable Haemophilus influenzae (NTHi) represents the cause of approximately one-third of episodes of OM. Current treatments for bacterial OM rely on the systemic use of antibiotics, which often leads to the emergence of multidrug resistant bacterial strains [6, 7]. Development of NTHi vaccine remains a challenge due to the high genetic diversity of NTHi strains and high antigenic variability of surface-exposed antigens [8, 9]. Therefore, there is an urgent need for developing alternate therapeutic strategies for treating NTHi infections. Previous studies have shown that NTHi upregulates MUC5AC transcription via Toll-like receptor- (TLR-) dependent activation of p38 MAPK and transcription factor AP-1 [10]. Due to the involvement of p38 MAPK in multiple cellular processes, therapies inhibiting it can have detrimental effects in the long-term. Thus identification of novel therapeutic strategies with minimal side effects is strongly desired.

Curcumin, a yellow pigment derived from the rhizome Curcuma longa, is reported to possess a broad range of pharmacological effects, including antioxidant, antitumor, anti-inflammatory, antimicrobial, and antidiabetic properties [11]. Curcumin does not present a dose-limiting toxicity, thereby potentiating long-term usage with minimal side effects [12]. Curcumin is classified as “generally recognized as safe (GRAS)” by the United States Food and Drug Administration. Despite its pleiotropic effects on a multitude of diseases, poor bioavailability presents a major limitation for curcumin usage [12]. We recently reported the inhibitory effect of curcumin on NTHi-induced neutrophil recruitment in a mouse model of OM [13]. However, the effect of curcumin on regulating MUC5AC mucin, a major contributor of OM pathology, remains to be evaluated.

2.3. Bacterial Strains and Culture Conditions

Clinical isolates of NTHi strains 12, 2627, and 9274 were used for this study [14, 15]. NTHi was prepared as described previously [13]. For in vitro experiments NTHi was resuspended in DMEM and used at a multiplicity of infection (MOI) of 50. For in vivo experiments, NTHi was resuspended in isotonic saline and used at a concentration of CFU per mouse. For inhibition studies, cells were pretreated with curcumin for 1 h prior to NTHi stimulation. For posttreatment studies, cells were treated with curcumin 1 h after NTHi stimulation.

2.4. Plasmids, Transfection, and Luciferase Assay

The expression plasmids, for constitutively active (CA) forms of MKK3 (MKK3b (E)) and MKK6 (MKK6b (E)) and dominant-negative mutant forms of p38α (fp38α (AF)) and p38β (fp38β2 (AF)), have been described previously [16]. MUC5AC-Luc luciferase reporter vector and AP-1 mutants of MUC5AC-Luc luciferase reporter vectors have been described previously [17]. Myc-MKP-1 overexpression plasmid has been described previously [13]. All transient transfections were performed using TransIT-LT-2020 transfection reagent (Mirus) according to the manufacturer’s protocol. Cells were assayed 48 h after transfection. Empty vector was transfected as a control. pRL-Renilla luciferase vector was from Promega. Luciferase activity was measured using Dual-Luciferase Reporter Assay System (Promega). MUC5AC luciferase activity was normalized to Renilla activity.

2.5. RNA-Mediated Interference

The human pSUPER-shMKP-1 knockdown construct as described previously [18] was transfected using TransIT-LT-2020 transfection reagent (Mirus) according to manufacturer’s protocol. Cells were assayed 48 h after transfection.

2.6. Real-Time Quantitative PCR (Q-PCR) Analysis

Total RNA was extracted with TRIzol reagent, according to manufacturer’s protocol (Life Technologies). TaqMan reverse transcription reagents were used to perform reverse transcription reaction (Applied Biosystems). Real-time quantitative PCR reactions were performed using Fast SYBR Green Master Mix (Applied Biosystems) and amplified and quantified with StepOnePlus Real-Time PCR system (Applied Biosystems). Relative quantities of mRNAs were calculated using the comparative Ct method and were normalized to control, human Cyclophilin or mouse glyceraldehyde-3-phosphate (GAPDH). Human (h) and mouse (m) primer sequences for hMUC5AC, hMKP-1, hCyclophilin, mMUC5AC, and mGAPDH primer sequences were previously described [18].

2.7. Enzyme-Linked Immunosorbent Assay (ELISA)

HMEECs were stimulated with NTHi for 12 h. Cell culture media were harvested and centrifuged at 12,000 ×g for 10 min to precipitate cell debris. Supernatants were assayed by direct ELISA method as described previously [19]. OD was measured using Benchmark Plus microplate spectrophotometer. MUC5AC protein concentration in the supernatant was determined by normalizing to the control group.

2.9. Mice and Animal Experiments

C57BL/6 mice (Jackson Laboratories) were employed. Anesthetized mice were inoculated with NTHi via the transtympanic route. For inhibition studies, mice were injected intraperitoneally (i.p) with curcumin (50 mg/kg) 1 h prior to or 1 h after NTHi inoculation. Total RNA was extracted from the dissected mice middle ear. All animal experiments were carried out following the guidelines approved by The Institutional Animal Care and Use Committee at Georgia State University.

2.11. Statistical Analysis

All experiments were repeated at least three independent times. Data are represented as mean standard deviation (s.d.). Statistical significance was assessed with unpaired student’s -test for data with two conditions () and ANOVA (followed by Tukey’s post hoc) for data with more than two conditions (), using SPSS 22 statistics software (IBM). was considered statistically significant.

Next, we sought to evaluate the therapeutic relevance of the inhibitory effect of curcumin on MUC5AC mucin in an NTHi-induced OM setting. Thus, we evaluated the effect of administering curcumin after NTHi infection, which resembles a clinically relevant setting. Curcumin administration after NTHi infection significantly suppressed NTHi-induced MUC5AC expression at mRNA (Figure 4(a)) and protein levels (Figure 4(b)) in vitro. Consistent with in vitro findings, curcumin treatment after NTHi infection inhibited MUC5AC mRNA (Figure 4(c)) and protein expression (Figure 4(d)) in mouse model for OM. Curcumin suppressed MUC5AC expression to the same extent under both pre-NTHi and post-NTHi infection conditions. Thus, these data suggest that curcumin is a potential therapeutic for treating NTHi-induced MUC5AC mucin overproduction as seen in OM.

Here we show that curcumin exerts its anti-inflammatory effects by increasing MKP-1 transcription. Increased MKP-1 transcription thereby enhances MKP-1 protein levels, which leads to p38 MAPK inhibition. Further studies are needed to identify the transcriptional factors, transcriptional machinery involved in curcumin-mediated MKP-1 induction. Previously it has been demonstrated that curcumin enhanced the phosphorylation and activation of MKP-1, leading to p38 MAPK inhibition [21]. Therefore, it is likely that curcumin modulates the activity of negative regulator MKP-1 via transcriptional, translational, and posttranslational mechanisms. Understanding the specific roles of these modulations and the specific signaling molecules involved could help identify novel drug targets for curcumin to counteract overactive immune responses.

In the current study curcumin was administered via intraperitoneal (i.p) injection. Systemic oral administration of antibiotics is the most common clinical strategy for treating OM. It is likely that systemic i.p administration of curcumin could have exerted global effects on the body including the middle ear. Our previous studies evaluating the effect of inhibitors such as rolipram, roflumilast, and vinpocetine on mucin expression in bacteria-induced OM model have also used i.p injection as the route of administration [18, 19]. Since curcumin is known for its safety and tolerability in patients, even with prolonged usage, it is likely that the systemic effects of curcumin would not be detrimental. However, to design therapies with increased specificity further studies focusing on evaluating the therapeutic efficiency of ototopical application of curcumin in resolving OM are needed. Further studies targeted towards increasing the bioavailability and development of ototopical drug delivery systems will be of clinical significance in treating OM. The insights of this study may have broader applications in the context of other chronic inflammatory conditions.

4. Conclusions

In summary, our study demonstrates that curcumin inhibits NTHi-induced MUC5AC expression in vitro and in vivo. Curcumin suppressed NTHi-induced MUC5AC expression by inhibition of p38 MAPK via upregulation of MKP-1 (Figure 4(e)). Curcumin treatment after NTHi infection also inhibited MUC5AC expression in a mouse model of OM, suggesting the clinical relevance of our findings. Thus, our study reports for the first time the efficacy of curcumin in treating NTHi-induced MUC5AC mucin overproduction.

Acknowledgments

This work was supported in part by National Institutes of Health Grants DC013833, DC005843, DC004562, and GM107529 (to Jian-Dong Li). Jian-Dong Li is Georgia Research Alliance Eminent Scholar in Inflammation and Immunity.